Transgenic, non-human animals can be used to understand the action of a single gene or genes in the context of the whole animal and the interrelated phenomena of gene activation, expression, and interaction. The technology has also led to the production of models for various diseases in humans and other animals which contributes significantly to an increased understanding of genetic mechanisms and of genes associated with specific diseases.
Traditionally, smaller animals such as mice have been used as disease models for human diseases and have been found to be suitable as models for certain diseases. However, their value as animal models for many human diseases is quite limited due to differences in mice compared to humans. Larger transgenic animals are much more suitable than mice for the study of many of the effects and treatments of most human diseases because of their greater similarity to humans in many aspects. Particularly, pigs are believed to be valuable as disease models for human diseases.
Atherosclerosis is by far the most frequent cause of coronary artery disease (angina pectoris, myocardial infarction and sudden death), carotid artery disease (stroke), and peripheral arterial disease. Atherosclerosis is referred to as ‘hardening of the arteries’ which is caused by the formation of numerous plaques within the arteries.
It is a chronic inflammatory disease, fueled by high plasma levels of cholesterol-rich lipoproteins, that leads to the development of atherosclerotic plaques of inflammatory cells, debris, and smooth muscle cells in large and medium-sized arteries1. These lesions by themselves rarely cause symptoms. The mechanical process wherein plaques burst open, known as plaque ruptures, causes the devastating consequences of atherosclerosis. By this process the thrombogenic core of the plaque is exposed to the haemostatic system of the circulating blood and this may elicit an acute flow-limiting superimposed thrombus.
In most cases of atherosclerosis the genetic component is complex, but in some cases the inheritance of the disease is monogenic. These cases are mostly due to mutations in genes coding for proteins involved in lipoprotein trafficking, and the most severe in humans are caused by homozygous null mutations in the low-density lipoprotein (LDL) receptor (homozygous familial hypercholesterolemia). Even though the disease process in homozygous LDL 10 receptor deficiency is immensely aggressive leading to severe coronary atherosclerosis in childhood, the disease is deemed qualitatively no different from that seen in more slowly developing atherosclerosis. Thus, monogenic causes of atherosclerosis can be used as tools to model atherosclerosis and atherosclerotic complications in genetically modified animal models. Apolipoprotein E (ApoE) and LDL-receptor deficient mice with severe hypercholesterolemia and rapid development of atherosclerosis were created in the early 1990s by homologous recombination in embryonic stem cells6,7. These mouse models have been instrumental in understanding many aspects of plaque development, but they are limited as models for human atherosclerosis because they lack measurable coronary atherosclerosis and do not develop the most feared complication of atherosclerosis, i.e. atherosclerotic plaque rupture and superimposed thrombosis. In addition—because of their small size—these animals have not aided research on bioimaging of atherosclerosis and percutaneous coronary intervention.
Similarities in cardiovascular structure and coronary artery distribution with humans make swine an attractive species to explore cardiovascular function and diseases. On conventional diets (˜3% fat, w/w), pigs have low plasma cholesterol levels (˜2 mmol/l), but many strains of pigs are susceptible to hypercholesterolemia and moderate atherosclerosis when fed a diet high in saturated fat and cholesterol, including miniature Yucatan8 and Yorkshire farm pigs9. Yucatan minipigs are of particular interest as models of human atherosclerosis because their adult weight compares well with that of humans (60-80 kg for males and 50-70 kg for females10) and thus equipment for imaging and percutaneous coronary intervention can be used directly.
The most pronounced lesions to date have been described in a progeny of large farm pigs identified in Wisconsin that harbor a single-nucleotide missense mutation in the LDL receptor gene that reduces affinity of the receptor to its ligands11. A colony of these pigs is now maintained in France by Professor Ludovic Drouet, INRA, Jouy en Josas. The pigs develop atherosclerosis in coronary arteries with many aspects of human atherosclerosis including plaque ruptures and superimposed thrombosis. However, hypercholesterolemia is modest on a normal pig diet (total cholesterol 5-8 mM) and atherosclerosis develops only slowly over several years. By the time these pigs have developed atherosclerosis, they are by far too large for most scientific purposes.
Even though considerable advances in anti-atherosclerotic pharmacological therapy have been achieved in the past decades, atherosclerosis remains one of the leading causes of death and severe disability in Denmark and worldwide2. There are at least three parts to an explanation for that.
First, the conventional population-based risk factor approach recommended in official guidelines is unable to identify those who need treatment on the level of the individual3. Thus, even though we have access to effective preventive treatment we are unable to identify those to treat. This problem could be solved by diagnostic imaging of atherosclerosis, which is becoming theoretically possible with the advent of new high-resolution imaging technology4,5. However, to develop tracers/contrast agents and imaging sequences that are able to visualize atherosclerotic plaques and atherosclerotic disease activity, we need a human-sized animal model of the disease that can be examined in patient CT, MR and PET-scanners.
Second, anti-atherosclerotic therapy is effective in preventing atherosclerosis in the long-term, but there is a lack of medical therapy that is effective at rapidly decreasing the risk of thrombotic complications in persons that have established severe atherosclerosis. E.g. in those persons that have identified themselves by presenting symptoms of atherosclerosis and in which maximal treatment is instigated, future events might still occur. Today, the major obstacle of developing such medicine is the lack of an animal model in which plaque rupture and arterial thrombosis occurs.
Third, the best treatment for coronary events today is primary percutaneous coronary intervention with placement of a stent, but these procedures are subject to complications including stent thrombosis and in-stent restenosis. Most research within this important area is carried out in non-diseased coronary pig arteries, but this approach has obvious limitations.
For these reasons, a human-sized pig model with severe human-like atherosclerosis, including plaque ruptures and thrombotic complications, is needed more than ever.
Even though the genes responsible for inherited atherosclerosis or involved in the development of disease have been identified in humans it does not follow that animals transgenic for such mutations display a phenotype comparable to that of the human disease. However, the present invention has surprisingly shown that the genetically modified pig models according of the present invention display the atherosclerosis phenotype.